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DESIGNING PRIMER FOR RAPID DETECTION OF POLLUTED RIVER WATER
RABI ATUN ADAWIAH BINTI ABDULLAH
UNIVERSITI TEKNOLOGI MALAYSIA
DESIGNING PRIMER FOR RAPID DETECTION OF POLLUTED RIVER WATER
RABI ATUN ADAWIAH BINTI ABDULLAH
A dissertation submitted in partial fulfillment of the
requirements for the award of the degree of
Master of Science (Biotechnology)
Faculty of Biosciences and Medical Engineering
Universiti Teknologi Malaysia
JANUARY 2013
ACKNOWLEDGEMENTS
Alhamdulillah all praises to The Almighty Allah . I would like to express my
deepest gratitude to:-
1. Dr. Adibah Binti Yahya for giving me chances to learn a lot through her guidance
and supervision.
2. Dr. Topik Hidayat (currently in Universiti Pendidikan Indonesia) for being awhile
and believing in me to carry on the project.
3. All lecturers and FBB (formerly known) staffs, guiding us a lot from the beginning.
4. Mr. Arman Bin Abdullah from FCE for helping me with bacteria genomic DNA
extraction.
5. Nurul Asmak, Amsal, Shahkila, Norfadilah, Qistina, Khoirunnisa, Amira Suriaty,
Nurain, Sadik, Noosha and the list go on of all members MQT batch 2012/2013.
6. Mr. Abdullah Bin Salleh and Pn. Samsiah Binti Abdullah, my beloved parents for
being my backbone through thick and thin in life.
7. Mohd Noor Haidzir Bin Mohd Fauzi, patiently cope with me and shows fully
support in achieving the dreams.
8. Nur Adani Aqmar, Muhammad Yusoff Qastalani, Nur Alifah Qistina, Siti Hajar
Fatimah and Hanis Maryam Aisyah, my beloved siblings for this would inspire them
in tertiary level education.
Last but not least, thank you to everyone either directly or indirectly whose I‟m in
contact with during my project. May all of them in bless of Allah. Thank you all.
ABSTRACT
Metagenomic now has been introduced as one of the new approaches to study
the community of uncultured bacteria. Furthermore, the findings of previous studies can
be stipulated to assess river water quality. Designing primer is the main focus in this
experiment to detect whether the water samples are polluted or unpolluted. The 16S
rRNA gene sequences of bacteria from previous studies have been used in the primer
designed. Previous findings show that two groups of bacteria have being classed to
polluted and unpolluted group of water samples. In this study, four water samples have
been collected from UTM Lake, Sg. Melana, Sg. Skudai and Sg. Kempas. The
unexpected findings show that primer sequences from unpolluted water samples has the
potential to prime coliform bacteria compared to primer sequences from polluted water
samples. The primers also being tested with wild types bacteria of E.coli, S.aureus,
Enterobacter sp. and also B.licherformis. The results also shown that primers designed
from unpolluted group can prime the sequences of the coliform wild type bacteria.
Many factors can contribute to the present results but the most common reason is DNA
extraction of the metagenome as previously being discussed by other researchers. This is
because most common bacteria in the freshwater are Gram positive bacteria and always
being underrepresented in the PCR library. Besides, different effluent or pollutants
compared to previous studies can have major effects to the genomic DNA extracted.
Further studies should be done to firmly state that the water is polluted or not only by
using specifically designed primer that can broadly prime bacteria in polluted water area
and also another specific primer sequences for unpolluted water.
ABSTRAK
Metagenomik telah menjadi salah satu pendekatan bagi mengkaji komuniti
bakteria yang tidak dapat dikultur dalam media di makmal. Tambahan pula, kajian yang
telah dilakukan sebelum ini mendapati metagenomik dapat digunakan untuk menilai
kualiti air. Fokus utama projek ini ialah dalam mereka jujukan primer bagi digunakan
untuk menentukan sampel air tersebut tercemar atau tidak tercemar. Sampel air di empat
kawasan sekitar Johor Bahru iaitu Tasik UTM, Sg. Melana, Sg. Skudai dan Sg. Kempas
diambil bagi diujikaji. Dapatan kajian menunjukkan jujukan primer daripada kumpulan
bakteria air tidak tercemar berpotensi mengesan bakteria koliform bagi sampel air
berbanding jujukan primer daripada kumpulan bakteria air tercemar. Hal ini boleh
disebabkan oleh beberapa faktor terutamanya ketika pengekstrakan DNA bakteria
seperti yang telah dialami oleh pengkaji-pengkaji sebelum ini. Hasil penemuan ujikaji
sebelum ini menyatakan bahawa bakteria Gram positif merupakan kumpulan bakteria
yang dominan bagi air tawar tetapi sentiasa tidak dipresentasi dengan baik dalam PCR
library. Selain itu, effluen atau bahan pencemar yang berbeza mengotori sumber air
yang sama boleh memberi kesan kepada dapatan kajian berbeza daripada sebelum ini.
Kajian selanjutnya perlu dilakukan supaya jujukan primer yang lebih spesifik dapat
direka untuk mengelaskan kualiti air sama ada tercemar atau tidak tercemar dengan
menggunakan kaedah ini. Oleh itu, jujukan primer tersebut dapat mengesan lebih
banyak bakteria bagi sumber air tersebut untuk ketepatan pengelasan yang lebih jitu dan
konsisten.
TABLE OF CONTENT
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xii
LIST OF FIGURES xiv
LIST OF SYMBOLS/ABBREVIATIONS xix
LIST OF APPENDICES xxi
1 INTRODUCTION 1
1.1 Study background 1
1.2 Problem statement 3
1.3 Objectives of study 4
1.4 Scope of study 4
1.5 Significance of study 5
2 LITERATURE REVIEW 6
2.1 Rivers as Freshwater 6
2.1.1 River pollution 8
2.1.2 Water Quality Assessment 10
2.2 Bacteria as bioindicator 10
2.3 Metagenomic 12
2.4 16S rRNA 13
2.5 Bioinformatics 15
3 METHODOLOGY 17
3.1 Experimental design 17
3.2 In-silico (Designing primers) 18
3.3 In-vivo (Sampling of water samples) 18
3.4 In-vitro (DNA extraction of bacteria metagenomic) 19
3.4.1 Gel electrophoresis 21
3.4.2 Polymerase chain reaction (PCR) 22
3.4.3 Culture of coliform bacteria 24
4 RESULTS AND DISCUSSIONS 26
4.1 Designing primers 26
4.2 Genomic DNA extraction 33
4.3 16S rRNA gene amplification by PCR 38
5 CONCLUSIONS 44
5.1 Conclusion 44
5.2 Recommendations 45
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Classification of River (Department of Environmental,
2011).
6
3.1 PCR master mix of 16S rRNA gene amplification. 22
3.2 PCR profile for amplification of 16S rRNA gene of
bacteria.
23
4.1 Designed primers of Prw and NPrw.
27
4.2
4.3
Summary of candidate primers based on Oligo Calc.
List of microorganisms retrieved from NCBI BLAST
from 16S rRNA database and nucleotide database.
28
30
.
LIST OF FIGURES
FIGURE NO. TITLE PAGE
4.1 Phenetic tree which derived from 24 isolates (18 clones
from polluted rivers; SY, SK and KM, and six clones
from non-polluted source, CN (Topik et al., 2012)
29
4.2 Genomic DNA from UTM Lake and Sg. Melana.
34
4.3 Genomic DNA from Sg. Kempas and Sg. Skudai water
samples.
35
4.4 Genomic DNA of wild type bacteria; 1) Escherichia
coli, 2) Staphylococcus aureus, 3) Enterobacter sp. and
4) Bacillus licherformis.
36
4.5 PCR products of UTM Lake and Sg. Melana.
39
4.6
4.7
PCR products of Staphylococcus aureus, Enterobacter
sp. Bacillus licherformis.
PCR product of Escherichia coli.
40
41
LIST OF SYMBOLS/ABBREVIATIONS
µl
L
0C
%
xg
NH3-N
UTM
DNA
rRNA/rDNA
Sg.
DOE
NWQS
WQI
WQS
BOD
COD
SS
DO
CTAB
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Microliter
Liter
Degrees Celsius
Percentage
Times gravity
Ammoniacal Nitrogen
Universiti Teknologi Malaysia
Deoxyribonucleic acid
Ribosomal Ribonucleic/Deoxyribonucleic acid
River (Sungai)
Department of Environment
National Water Quality Status
Water Quality Index
Water Quality Status
Biological Oxygen Demand
Chemical Oxygen demand
Suspended Solid
Dissolved Oxygen
Cetyltrimethyl ammonium bromide
PVP
bp
kb
PCR
Zn
Cu
Ni
Cr
As
Pb
Hg
Cd
-
-
-
-
-
-
-
-
-
-
-
-
Polyvinylpyrrolidone
Basepair
Kilo basepair
Polymerase Chain Reaction
Zinc
Copper
Nitrogen
Chromium
Astatine
Lead
Mercury
Cadmium
LIST OF APPENDICES
APPENDIX TITLE PAGE
A
Preparation of CTAB buffer 54
B
Aqueous phase : 0.08 Ammonium acetate : 0.54
Isopropanol
55
C Ingredients of Solutions 56
D Nucleotide sequences from PCR cloned library 58
CHAPTER 1
INTRODUCTION
1.1 Study background
River is one of the water resources on earth besides salt water; seas and oceans
and also other freshwater; lakes and underground water. As studied by Das and Acharya
(2003), three quarters of planet earth‟s surface is covered by salt water and another
quarter is the land that holds the freshwater including rivers. Ironically, river water
pollution has been worse years by years even though humans are developing means to
green world. It is very crucial to prevent this from being much worse as the water that
we have been using daily in our homes, schools, shops, offices, factories and in many
activities comes from rivers.
The later product of water usage is the sewage or waste water that needs proper
treatment before being released to municipal water sources. This is because, without
proper sewage treatment the river will be polluted with effluents and runs off from
factories waste water, other domestic sewage and also litter thrown into the river and
cause many bad effects not only to human health but also to other organisms especially
aquatic animals and plants.
River water pollution has high effect to water quality either for the sake of
humans, aquatic animals and plants. Humans rely on river waters for drinking needs,
foods, agriculture, transportation and many more besides being habitats for aquatic
animals and plants which is to ensure ecological balance. The key to understand water
quality is to understand what is „living‟ beneath it other than aquatic animals and plants
which is the microbial community. These are because microbes are ubiquitous and the
consumption of organic and inorganic matter depends on unique microbial metabolism
usually involves the enzymatic activities for the survival in polluted or stress
environment (John et al., 2010). And one way to understand the river conditions are by
understanding the microbe‟s genome.
Metagenomic studies known for studying many species of microorganisms as a
whole instead of as individual in the laboratory. Metagenomic is also referred to as
"community genomics" or "environmental genomics" because it is the sequencing and
analysis of DNA of microorganisms recovered from the environment directly without
the need for culturing the microorganisms (John et al., 2010). According to Cottrell et
al., (2005) phylogenetic information is needed together with estimates of metabolic
potential in order to link specific members of the community to biogeochemical
processes. One approach to obtain this phylogenetic information is to screen
metagenomic libraries for 16S rRNA genes in order to identify clones that can be used
to explore the metabolic potential of targeted bacterial groups (Beja et al., 2001; Quaiser
et al., 2002; Liles et al., 2003). 16S rNA gene sequence has hindered the technical
difficulty of obtaining their sequences as opposed to clonal cultures without isolation of
16S rRNA or cloning of its gene into a host such as E.coli (Lane et al., 1985). This
method also simplified a rapid expansion of 16S rRNA sequences collection available
for phylogenetic analyses.
1.2 Problem statement
The atmosphere, rivers, lakes and underground hold less than 1% of all the fresh
water and this tiny amount has to provide the fresh water needed to support the earth's
population. With the increased population will also increase the water needed to supply
sufficiently. Fresh water is a precious resource and the increasing pollution of our rivers
and lakes have causes an alarm to find alternative approach to deal with it.
Microorganisms have unique ways to live in polluted environment. Thus, the presence
of particular microbes in the environment may indicate the carbon source or types of
conditions they lived in. By manipulating the mass number of the microbes, we can use
it to study the environment particularly in river water either it is polluted or non-polluted
by studying which microbes present in either both conditions. Furthermore, the
presences of coliform microorganisms which are disease-causing bacteria in polluted
water not only affect the environment, but also increase health problems in humans and
other living organisms if consumed. Thus, by using molecular techniques to assess the
river quality, biological methods not only provide the results of pollution level but also
the microorganisms beneath it that can indicates types of pollutants in polluted river
water. In addition with combining physical and chemical methods, it will confirm other
biochemical tests which are complex in just one Polymerase Chain Reaction (PCR) test
with the primers designed in this experiment particularly for river water in Malaysia for
the time being. These will help the authorities a lot to conduct screening method in just a
few hours compared to other tests which take longer time up to five days and above.
1.3 Objectives of study
The objectives are:-
I. To design primers from the cloned library of DNA obtained from
polluted and non-polluted river water.
II. To select for a good candidate of designed primers using in-silico public
access software: Oligo Calc and Oligo Analyzer.
III. To test good candidate of designed primers in-vitro using environmental
samples.
IV. To confirm bacteria as bioindicator for river water assessment.
1.4 Scope of study
The main focus of this study is designing universal primer by using
bioinformatics tools to be used for rapid detection of polluted river water. As there is
less findings of biological screening by using molecular approach for river water
assessment particularly in Malaysia, this preliminary experiment in studying
metagenomic field will hopefully be beneficial to effectively assess the water quality.
Furthermore, it is simpler, easier, less time consumed and good results produced
compared to conventional methods. Topik et al., (2012) in his previous study, already
did with PCR clone library that emphasizes the major groups of microbes from polluted
and non-polluted river water. The screening part provides useful information for
designing primer in this experiment where it was tested on environmental water
samples.
1.5 Significance of study
This study is aimed at achieving the main goal for rapid detection of detecting
river water pollution by using molecular techniques and bioinformatics tools (designed
primers) as an alternative to the conventional method such as physical and chemical
methods of BOD and COD. This is because, only a small percentage of microorganisms
in nature can be cultured which means that the extant genomic data are highly biased
and do not present the true picture of genomes of microbial species (Amann et al., 1995;
Pace., 1997; Rappe and Giovannoni., 2003). Thus it is important to know the best way
to assess the quality of river water. The best way will give the results either the river
water has been polluted or not in a short time and also details of water content to
recognize the pollutants for further actions to be taken. Metagenomic method which
process usually takes only up to 24 hours will exclude the laborious culturing method.
On the other hand, metagenomic also allow a more accurate elucidation of microbial
population study as it can include the molecular information of bacteria which are very
difficult to be cultured in the lab or also known as unculturable bacteria. The simplest
screening approach by using good candidate designed primers to assess water quality
had been done in this study that excludes the limitations of unculturable bacteria. The
findings of this experiment will enable bacteria being used widely as bio-indicator to
assess water quality as suggested by Topik et al. (2012) whereby less laborious, less
time consumed and cost-effective approach had been applied by using bio-informatic
tools and PCR to imply these simplest water assessment screening approach.
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